Patent Application
20080057593
The present invention relates to the prediction, diagnosis and differential diagnosis of Alzheimer's disease. More particularly, the present invention provides a method to determine whether a subject, that does not show any clinical signs of Alzheimer's disease, has a likelihood to develop Alzheimer's disease. The present invention further provides a method for the diagnosis of AD and/or for the differential diagnosis of Alzheimer's disease versus other dementias such as dementia with Lewy bodies.
Dementia is a serious, common, and rapidly growing worldwide problem associated with increased healthcare utilization. It is a major predictor of morbidity and mortality in the elderly. The occurrence of the more than 100 known diseases that produce this condition depends on age, as well as genetic factors linked to geography, race, and ethnicity. Dementia can be defined as a chronic deterioration in multiple cognitive abilities (memory, attention, judgment, etc.) that impairs the previously successful performance of activities of daily living. Its clinical profile and degree of severity are affected not only by the total quantity of neuronal loss, but by the specific locations of the underlying lesions. By far the most common forms of dementia are Alzheimer's disease (40-60% of the cases), dementia with Lewy bodies (10-20% of the cases), vascular dementia (25% and possibly contributing in up to 40% of the cases), and frontotemporal dementia (for which prevalence remains unclear) (Lowe, 2001; Leys et al., 2002; McKeith, 2002; Knopman et al., 2002). More than 33% of women and 20% of men over the age of 65 will develop dementia or milder forms of cognitive impairment in their lifetime (Yaffe and Gregg, 2002).
Alzheimer's disease (AD), the principle form and prototype of dementia, may be classified according to different criteria. From the genetic point of view, the disease can be categorized into two types: (i) less frequent, inherited familial forms (ranging from <5% for early-onset to 10-15% for late-onset forms when all genetic predisposition factors are included), and (ii) the far more common sporadic type for which no obvious inheritance patterns have been established. The sporadic form generally emerges after 65 years of age, and is thought to be multifactorial in nature.
The definitive diagnosis of AD is based on the finding of disruptively large amounts of senile plaques and neurofibrillary tangles in the affected areas of the neocortex at autopsy. Along with massive gray matter atrophy, these two types of abnormal structures are the hallmarks of the disease.
Neurofibrillary tangles consist of abnormal collections of twisted threads found inside the nerve cells. The chief components of these tangles are abnormal aggregations of a protein called tau. In the central nervous system, normal tau proteins bind and stabilize microtubules that are key constituents of the cell's internal structure. In AD, however, tau is hyperphosphorylated and twists itself into paired helical filaments: two threads of tau wound around each other. These filaments aggregate to form the telltale neurofibrillary tangles (Goedert, 1996).
The second hallmark of AD, senile plaques, consist largely of an insoluble peptide called β-amyloid (Aβ) that is surrounded by a variety of neuronal and glial processes. This amorphous, acellular material is found in the spaces between the brain's nerve cells. Aβ is a small peptide found mainly in two sizes, 40 (Aβ40) and 42 (Aβ42) amino acids, and in minor amounts in other sizes (see further). Aβ is known to be metabolised from the proteolytic cleavage of APP (Saido, 2000) along two pathways. In the first non-pathogenic route, the cleavage of the APP molecule by secretase enzymes leads to shorter non-amyloidogenic proteins (Aβ39 or Aβ40). In the second disease-related pathway, the APP cleavage yields the longer and potentially amyloidogenic Aβ42 fragment that tends to misfold and aggregate into polymer chains that not only seed the plaques, but may ultimately cause neuronal damage (Selkoe, 1991).
Amyloid found in senile plaque cores is primarily Aβ42 (Roher et al., 1993; Miller et al., 1993). Amyloid deposits are sparsely found in different regions of the normal aging brain, but become increasingly more abundant in the initial and subsequent stages of Alzheimer's disease. Secreted soluble Aβ is a product of normal cell metabolism and is found in various body fluids including plasma and CSF. In biological fluids, Aβ40 and Aβ42 are generally believed to be the major amyloid species (Seubert et al., 1992; Shoji et al., 1992; Vigo-Pelfrey et al., 1993; Ida et al., 1996). In addition to Aβ40 and Aβ42, however, Aβ37/38/39 has also been found in CSF from AD and normal subjects (Wiltfang et al., 2002).
The N-terminus is the most heterogeneous part of Aβ, being subject to truncation, racemization and isomerization. Since the discovery of Aβ as the major constituent of amyloid deposits in AD (Glenner and Wong, 1984), different N-terminally truncated and/or modified Aβ peptides have been identified in plaques of AD patients (Masters et al., 1985; Mori et al., 1992; Näslund et al., 1994; Saido et al., 1995; 1996; Iwatsubo et al., 1996; Russo et al., 1997; Tekirian et al., 1998; Larner, 1999; Thal et al., 1999, Harigaya et al., 2000; Wiltfang et al., 2001; Kalback et al., 2002; Sergeant et al., 2003). N-terminally truncated and/or modified Aβ peptides were also identified in CSF of AD and normal subjects (Seubert et al., 1992; Vigo-Pelfrey et al., 1993; Ida et al., 1996; Lewczuk et al., 2003; 2004).
The second most common cause of primary dementia, after Alzheimer's disease, is dementia with Lewy bodies (DLB) accounting for no less than 10% to 20% of all cases of dementia (Lowe, 2001; McKeith, 2002). The disease-defining Lewy bodies are neuronal inclusions composed of abnormally phosphorylated neurofilaments, ubiquitin, and alpha-synuclein. These abnormalities are thought to contribute to neurological dysfunction resulting in clinical symptoms which, depending on the brain region affected, may partially resemble those associated with Alzheimer's and Parkinson's disease. Indeed, many cases of DLB are still erroneously misdiagnosed as Alzheimer's disease. However, differentiation of DLB from Alzheimer's disease is important. This is because certain neuroleptic agents, extensively prescribed for the treatment of psychotic symptoms and behavioral disturbances common in dementia, may result in severe (potentially lethal) hypersensitivity reactions in the case of DLB (Mc Keith, 2002). In addition, the pathological mechanisms that cause DLB may be fundamentally different from those in AD and, accordingly, the differentiation of DLB from AD might be of relevance for disease-modifying treatment aimed at these pathological mechanisms.
Recent clinical research has identified a transitional state between the cognitive decline found in normal aging and dementia. This prodromal state has been termed mild cognitive impairment (MCI). Persons with MCI experience consistent memory deficits earlier and to a greater extent than one would expect at their age, but remain functionally independent. Therefore, they fall short of fulfilling the accepted criteria for a positive diagnosis of one of the primary dementias (Petersen et al., 2001). Individuals who present clinically mild cognitive symptoms belong to a heterogeneous group and may not share the same fate ultimately. Some may go on to develop AD, while others may progress to another form of dementia. It is possible that some of the subjects will never progress beyond the state of MCI. Nevertheless, depending on the cohort source and definition, between 19% and 50% of individuals diagnosed with MCI progress to dementia (usually Alzheimer's disease) (Chertkow, 2002).
A very significant effort is underway to test a large number of therapeutic options for AD and other dementias. These approaches include numerous agents such as acetycholinesterase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDS), estrogen, neurotrophic agents, and even vitamins (Sramek and Cutler, 2000; Thal, 2000) as well as the reduction of senile plaque formation by the identification of secretase inhibitors and the development of an immunization model for the prevention of amyloid deposition (Conde, 2002). Since disease modifying therapy is likely to be most effective early in the course of the disease, MCI might be the optimal stage for therapeutic intervention (Chertkow, 2002). While no treatments are recommended for MCI currently, clinical trials regarding potential therapies are under way. Early diagnosis is therefore highly desirable before neurodegeneration becomes severe and widespread. Therefore, for the physician, a key challenge is to assess whether the patient has crossed the thin line between normal aging and MCI. Indeed, incipient dementia must be clearly distinguished from benign memory problems associated with age, anxiety, lack of attention, co-existent medical problems, or depression. In addition, as some of the forms of dementia are partially or even completely reversible upon treatment (e.g. dementia due to vitamin deficiency, drug intoxication, alcoholism, endocrine disorders, etc.), the importance of timeliness and accuracy in clinical diagnosis and differential diagnosis is clear.
Diagnosis of dementias such as AD, is currently based on a broad, comprehensive work-up that consists of (i) a thorough clinical evaluation (incl. physical exam, anamnesis with patient and family, medication review); (ii) a neurological examination involving neuropsychological tests and radiology; and (iii) laboratory testing (e.g., vitamin B12, folic acid, thyroid function, complete blood chemistry and blood count, etc.) (Marin et al., 2002) and exclusion of all other forms of dementia. However, ultimately, only confirmation by autopsy can unequivocally differentiate between the various dementing disorders.
The value of neuropsychological tests to help identify the presence of early memory and cognitive impairments—and quantify the degree to which the patient is affected—has been well documented (Welsh et al., 1991; Petersen et al., 1994; Masur et al., 1994). However, in the very early stages of the disease, delineating disease process from “normal aging” remains difficult. Even in later stages of the disease, diagnosis of AD and distinguishing AD from a number of neurodegenerative diseases associated with dementia, and especially from DLB, may also be difficult.
Diagnostic procedures need to be improved so that they can identify AD at pre-dementia stage and differentiate AD from other causes of cognitive impairment or dementia. Some aspects of the pathological cascade of AD are reflected in altered protein concentrations in body fluids. Such proteins (biomarkers or biological markers) which reflect the central pathogenic processes associated with the disease, namely the degeneration of neurons and their synapses as reflected in their defining characteristic lesions—senile plaques and neurofibrillary tangles (The Ronald and Nancy Reagan Research Institute of the Alzheimer's Association of the National Institute on Aging Working Group, 1998), can add to the accuracy of this early and differential diagnosis. Since the entire brain is in direct contact with the CSF and AD and related disorders are considered as brain diseases, the chance of finding significant differences is likely to be in this body fluid.
Two such mainstream biomarkers are CSF-Aβ protein ending at amino acid 42 (Aβ42) and CSF-tau protein. For its part, the concentration of CSF-Aβ42 appears to be linked with the deposition of β-amyloid into extracellular senile plaques (Motter et al., 1995; Andreasen et al., 1999a). For CSF-tau, the levels of this protein are thought to reflect neuronal and axonal degeneration (Blennow et al., 1995; Andreasen et al., 1998) or the possible formation of neurofibrillary tangles (Tapiola et al., 1997). Significantly increased CSF-tau levels (Motter et al., 1995; Vigo-Pelfrey et al., 1995) and markedly reduced CSF-Aβ42 concentrations (Blennow et al., 1995; Tapiola et al., 1997) were both observed in patients with Alzheimer's disease compared to non-demented controls. Indeed, large, well-designed, multicenter studies in the United States (Galasko et al., 1998) and Japan (Kanai et al., 1998), and an international multicenter study (Hulstaert et al., 1999) consistently found that the combined use of CSF-tau and CSF-Aβ42 markers resulted in a high sensitivity and specificity, and met the requirements for discriminating Alzheimer's disease from normal aging and specific neurological disorders (Sunderland et al., 2003). This clearly places their joint use well within the consensus guidelines.
Another potential breakthrough for the improved diagnosis of dementia is linked to the observation of the relative absence of abnormally phosphorylated tau protein in the brain cells of patients with non-tau dementias (eg. Parkinsons's disease, DLB) versus the high amounts found in those with Alzheimer's disease (Harrington et al., 1994). Significant differences were shown in CSF-phospho-tau levels between Alzheimer's disease and other dementias, especially DLB (Pametti et al., 2001; Vanmechelen et al., 2001).
Wiltfang and coworkers (2001) also observed an elevated level of Aβ(2-42) in CSF from AD patients. Both CSF-Aβx-42(43) and Aβ1-42(43) levels were significantly lower in AD patients than in a control group, whereas neither CSF-Aβx-40 nor CSF-Aβ1-40 levels showed any differences between the two groups (Tamaoka et al., 1997). It was, however, not specified which Aβx-42 peptides were measured in this study.
In addition, none of the above studies assessed the behaviour of these biomarkers in cognitive impaired patients or their value in the prediction of progression of MCI patients to dementia.
Because memory impairment and MCI may be clinically and pathologically heterogeneous, biomarkers may be particularly useful for identifying subtypes. At present, there are no definite data on the usefulness of biomarkers in classifying MCI and predicting whether a patient with MCI will develop a primary dementia such as Alzheimer's disease. The measurement of CSF-tau might be used effectively for identifying incipient AD among patients diagnosed clinically as having MCI (Sunderland et al., 1999; Riemenschneider et al., 2002). However, Buerger et al. (2002) observed that high CSF-phospho-tau levels, but not CSF-tau levels correlated with cognitive decline and conversion from MCI to AD. Arai et al. (2000) observed elevated tau and phospho-tau in the CSF of patients with MCI who went on to develop AD. Okamura et al. (2002) used the CSF-CBF index to discriminate MCI that progressed to AD from MCI that did not progress to AD. The CSF-CBF index is based on CSF-tau levels divided by regional cerebral blood flow (CBF) in the posterior cingulate cortex. Several studies report on the use of CSF-Aβ42 (in combination with CSF-tau and/or CSF-phospho-tau) as a biological marker for MCI progressing to AD (Andreasen et al. 1999b; Riemenschneider et al., 2002; Andreasen et al., 2003). Non-demented individuals who developed dementia during a follow-up period of three years already had lower Aβ(1-42) levels (but not Aβ(1-40)) than those who remained non-demented (Skoog et al., 2003). The level of β-amyloid forms seems to decrease constantly from the onset of AD. Although the ratio Aβ40/Aβ42 showed an increase with the progression of AD, its increase already had started before the appearance of the clinical symptoms of AD (Kanai et al. 1998).
The above shows that certain biomarkers are abnormally altered before conversion to clinical dementia and they could be promising as potential early markers to identify MCI patients that will develop primary dementia (especially Alzheimer's disease).
Such findings point towards the future combined use of such biomarkers, together with other appropriate tests, as part of a broad diagnostic work-up for MCI, Alzheimer's disease, and other dementias. It is therefore of extreme importance that additional biomarkers are identified that are already altered in patients before their conversion to clinical dementia and that could aid in the diagnosis of AD and prediction of cognitive impaired patients that will progress to dementias such as AD.
The present invention provides methods and diagnostic kits for the prediction, diagnosis and differential diagnosis of Alzheimer's disease (AD). More particularly, the present invention provides a method and a diagnostic kit to determine whether a subject that at the time of sampling does not show any clinical signs of AD, has a likelihood to develop AD. The methods and diagnostic kits of the present invention are based on the determination of x/y ratios in body fluid samples obtained from the subjects under diagnosis whereby:
The x/y ratios in body fluid samples obtained from subjects that, at the time of sampling did not show any clinical signs of AD and that later developed AD, appeared to be significantly altered compared to the x/y ratios in body fluid samples obtained from subjects that at the time of sampling did not show any clinical signs of AD and that did not develop AD. Accordingly, the present invention provides a method and a diagnostic kit to determine whether a subject has a likelihood to develop AD, comprising the following steps:
The method and diagnostic kit of the present invention can be carried out on body fluid samples obtained from subjects that do not show any clinical signs of memory impairment, MCI, or dementia. In a preferred embodiment, the method of the present invention is carried out on body fluid samples obtained from subjects with memory impairment or subjects that are clinically diagnosed as having MCI.
The present invention also provides methods and diagnostic kits for the diagnosis of subjects suffering from AD and/or for the differential diagnosis of subjects suffering from AD versus subjects suffering from other dementias such as DLB. The methods and diagnostic kits of the present invention are based on the finding that the x/y ratios in a body fluid samples obtained from subjects suffering from AD are significantly altered compared to the x/y ratios in body fluid samples obtained from control subjects and subjects suffering from DLB. Accordingly, the present invention provides methods and diagnostic kits for the diagnosis of a subject suffering from AD and/or for the differential diagnosis of a subject suffering from AD versus a subject suffering from another dementia such as DLB, comprising the following steps:
In a preferred embodiment of the present invention, x is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid) of the Aβ peptide. Even more preferably, x is the level of Aβ peptides capable of forming an immunological complex with the monoclonal antibody 3D6, BAN-50, and/or Anti-N1(D). In another preferred embodiment of the present invention, y is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid) of the Aβ peptide. Even more preferably, y is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope different from the 3D6, BAN-50, and/or Anti-N1(D) epitope, such as the monoclonal antibody 4G8, the monoclonal antibody 6E10, and/or the monoclonal antibody 10H3. In another preferred embodiment, x is the level of Aβ(1-C) peptides and y is the level of Aβ(N-C) peptides. In another preferred embodiment, x is the level of Aβ(1-42) and/or Aβ(1-43) peptides and y is the level of Aβ(N-42) and/or Aβ(N-43) peptides. In another preferred embodiment, x is the level of Aβ(1-42) peptides and y is the level of Aβ(N-42) peptides.
The methods and diagnostic kits of the present invention can be used on any body fluid sample obtained from a subject. In a preferred embodiment, the body fluid sample is a cerebrospinal fluid sample or a plasma or serum sample.
The present invention provides methods for the prediction, diagnosis and differential diagnosis of AD. More particularly, the present invention relates to a method to determine whether a subject that does not show any clinical signs of AD has a likelihood to develop AD. The method of the invention comprises the following steps:
The present invention is based on the finding that this ratio x/y, as defined above, was significantly decreased in body fluid samples obtained from subjects that at the time of sampling did not show any clinical signs of AD and that later developed AD, compared to this x/y ratio in body fluid samples obtained from subjects that at the time of sampling did not show any clinical signs of AD and that did not develop AD. The indication that this ratio x/y is significantly altered in body fluid samples of subjects that will develop AD forms a basis for the development of a diagnostic test for determining whether a subject has a likelihood to develop AD. At present, delineating disease process from “normal aging” still remains difficult. Early diagnosis of a disease process, before clinical signs of dementia are present is, however, highly desirable, since disease modifying therapy is likely to be most effective early in the course of disease
The subject under diagnosis in the above method can be any subject that does not show clinical signs of dementia. The subject under diagnosis may be a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, a monkey, a rabbit, a hare, a chicken, a dog, a cat, a mouse, a rat, an elk, a deer, a tiger, a zebrafish, a pufferfish, a fly, a worm or C. elegans. More preferably, the subject is a primate. Even more preferably, the subject is a human. In a preferred embodiment, the subject is a human who does not show any clinical signs of AD according to the NINCDS-ADRDA criteria (McKhann et al., 1984), the ICD-10 criteria (World Health Organization, 1992), and/or the DSM-IV criteria (American Psychiatric Association, 1994). The term “AD” shall mean Alzheimer's disease.
The above method can be carried out on body fluid samples obtained from subjects that, in addition to the absence of clinical signs of dementia or AD, also do not show any clinical signs of memory impairment or MCI. In a preferred embodiment of the invention, however, the above method is carried out on body fluid samples obtained from subjects suffering from memory impairment or subjects that suffer from MCI. Clinical diagnosis of memory impairment and MCI is currently done according to Petersen et al. (1999), Palmer et al. (2003) and/or Wahlund et al. (2003).
In the present invention, the terms “develop AD”, “progress to AD”, “will have AD”, etc. are used interchangeably and mean that the subject at the time of sampling of the body fluid (on which the method of the present invention is carried out), does not show any clinical signs of AD, but that said subject shows clinical signs of AD within a period of maximum 5 years, preferably maximum 3 years, and most preferably within 1 year after the sampling of said body fluid.
The terms “likelihood”, “risk”, “susceptibility”, “predisposition”, “prognosis” or “prediction” are interchangeable and are used with respect to the probability of developing AD.
The present invention also relates to a method for the diagnosis of a subject suffering from AD and/or for the differential diagnosis of subjects suffering from AD versus subjects suffering from other dementias such as DLB. The method of the invention comprises the following steps:
The above method is based on the finding that the x/y ratios, as defined above, in body fluid samples obtained from subjects that suffer from AD are significantly decreased compared to the x/y ratios in body fluid samples obtained from control subjects and from subjects that suffer from DLB. The indication that the x/y ratio in body fluids samples obtained from subjects suffering from AD is significantly altered compared to the x/y ratio in body fluid samples obtained from control subjects and from subjects suffering from DLB forms a basis for the development of a diagnostic test for the diagnosis of AD and/or the differential diagnosis of AD versus other dementias such as DLB. The term “diagnosis” means that subjects suffering from a certain neurological disease are discriminated from subjects not suffering from said neurological disease. In the present invention, subjects suffering from AD are discriminated from control subjects. The term “differential diagnosis” means that subjects suffering from a certain neurological disease are discriminated from subjects suffering from another neurological disease. In the present invention, subjects suffering from AD are discriminated from subjects suffering from another dementia such as DLB. Although criteria for the diagnosis of AD (McKhann et al., 1984; World Health Organization, 1992; American Psychiatric Association, 1994) and other dementias such as DLB (McKeith et al., 1996) are currently available, they may lack sufficient detail to discriminate these dementias such as DLB from AD (McKeith, 2002). The differentiation of subjects suffering from AD from subjects suffering from DLB remains a major problem. Ultimately, only autopsy can unequivocally differentiate between the various dementing disorders. In view of potential differing therapeutic implications for subjects suffering from AD and subjects suffering from DLB, this differentiation is, however, of extreme importance. The method of the present invention provides an additional tool in the differential diagnosis of subjects suffering from AD versus subjects suffering from DLB. The term “DLB” shall mean dementia with Lewy bodies.
The subject under diagnosis in the above method can be any subject that shows clinical signs of dementia. The subject under diagnosis may be a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, a monkey, a rabbit, a hare, a dog, a cat, a mouse, a rat, an elk, a deer or a tiger. More preferably, the subject is a primate. Even more preferably, the subject is a human. Control subjects are subjects without histories, symptoms or signs of psychiatric or neurological disease.
The methods of the present invention are based on the detection of the ratio x/y in body fluid samples obtained from the subject under diagnosis. The term “body fluid” refers to all fluids that are present in the body including but not limited to blood, lymph, urine, and cerebrospinal fluid (CSF), containing Aβ peptides. The blood sample may be a plasma sample or a serum sample. It could be possible that x is determined in a certain body fluid sample while y is determined in another body fluid sample. In a preferred embodiment, however, x and y are determined in the same body fluid sample. In a preferred embodiment of the present invention the x/y ratio is determined in a cerebrospinal fluid sample taken from the subject. The term “cerebrospinal fluid” or “CSF” is intended to include whole cerebrospinal fluid or derivatives of fractions thereof well known to those skilled in the art. Thus, a cerebrospinal fluid sample can include various fractionated forms of cerebrospinal fluid or can include various diluents added to facilitate storage or processing in a particular assay. Such diluents are well known to those skilled in the art and include various buffers, preservatives, and the like.
Within the ratio x/y of the present invention, the numerator (x) is defined as the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. Within the ratio x/y of the present invention, the denominator (y) is defined as the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. Aβ peptide, Aβ, β-amyloid peptide, or A4 peptide are used interchangeably throughout the present invention and refer to a peptide of 37-43 amino acids (Aβ37, Aβ38, Aβ39, Aβ40, Aβ41, Aβ42 or Aβ43), which is the principal component of characteristic plaques of Alzheimer's disease. Aβ is generated by processing of a larger protein APP by two enzymes, termed β- and γ-secretases (
Aβ41, Aβ40, Aβ39, Aβ38 and Aβ37 differ from Aβ42 by the omission of Ala (A), Ile-Ala (IA), Val-Ile-Ala (VIA), Val-Val-Ile-Ala (VVIA) and Gly-Val-Val-Ile-Ala (GVVIA) respectively, from the C-terminal end. Aβ43 differs from Aβ42 by the presence of a threonine residue at the C-terminus. In the methods of the present invention Aβ peptides with any C-terminal ending are detected. In a preferred embodiment of the present invention, the level of Aβ peptides ending at Ala42 (Aβ42) and/or Thr43 (Aβ43) is determined. In another preferred embodiment, the level of Aβ peptides ending at Ala42 (Aβ42) is determined. The Aβ peptides that are detected in the methods of the present may also include isomerized peptides. Aspartic acid-bond isomerization has, for example, been described by Szandrei et al. (1996). The Aβ peptides that are detected in the methods of the present invention (x and y) are also termed “specific Aβ peptide” or “specific Aβ peptides” and refer to Aβ peptides capable of forming an immunological complex with an antibody that recognizes a certain epitope of the Aβ peptide. The term “capable of forming an immunological complex with”, “(specifically) recognizing”, “(specifically) binding with”, “(specifically) reacting with”, or “(specifically) forming an immunological reaction with” refers to a binding reaction by the antibody to the specific Aβ peptide which is determinative of the presence of said specific Aβ peptide in the sample in the presence of a heterogeneous population of other peptides, proteins, and/or other biologicals. Thus, under the designated immunoassay conditions, the specified antibody preferentially binds to the specific Aβ peptide, while binding to other peptides or proteins does not occur in significant amounts. The binding of an antibody to an Aβ peptide depends on the epitope available on said Aβ peptide. The term “epitope” refers to that portion of the antigen (i.e. the Aβ peptide) that is specifically bound by an antibody-combining site. Epitopes may be determined by any of the techniques known in the art or may be predicted by a variety of computer prediction models known in the art.
For the numerator of the ratio x/y (x), the epitope recognized by the antibody that is capable of binding with the detected “specific Aβ peptide” should contain the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. In a preferred embodiment, said epitope should contain the first amino acid (D; aspartic acid) of the Aβ peptide. Accordingly, in this preferred embodiment, x is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid) of the Aβ peptide. Examples of antibodies recognizing an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid) of the Aβ peptide include (but are not limited to) the monoclonal antibody 3D6, the monoclonal antibody BAN-50 and the monoclonal antibody Anti-N1(D) (Table 1). Accordingly, in a preferred embodiment, x is the level of Aβ peptides capable of forming an immunological complex with the monoclonal antibody 3D6, BAN-50, and/or Anti-N1(D).
For the denominator of the ratio x/y (y), the epitope recognized by the antibody that is capable of binding with the detected “specific Aβ peptide” may not contain the first amino acid (D, aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide.
In a preferred embodiment, for the numerator (x), the epitope recognized by the antibody that is capable of binding with the detected “specific Aβ peptide” contains the first amino acid (D; aspartic acid) of the Aβ peptide and, for the denominator (y), the epitope recognized by the antibody that is capable of binding with the detected “specific Aβ peptide” may not contain the first amino acid (D; aspartic acid) of the Aβ peptide. Accordingly, in this preferred embodiment, y is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid) of the Aβ peptide. Said epitope should thus be different from the 3D6, BAN-50, and/or Anti-N1(D) epitope and y is thus the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope different from the 3D6, BAN-50, and/or Anti-N1(D) epitope. Examples of such antibodies that recognize an epitope of the Aβ peptide that does not contain the first amino acid (D; aspartic acid) of the Aβ peptide include (but are not limited to) the antibodies as given in Table 1. Preferred antibodies include 4G8, 6E10, and 10H3. In accordance, in a preferred embodiment, y is the level of Aβ peptides capable of forming an immunological complex with the monoclonal antibody 4G8, with the monoclonal antibody 6E10, and/or with the monoclonal antibody 10H3.
In another embodiment, for the numerator (x), the epitope recognized by the antibody that is capable of binding with the detected “specific Aβ peptide” contains the second amino acid (A; alanine) of the Aβ peptide and, for the denominator (y), the epitope recognized by the antibody that is capable of binding with the detected “specific Aβ peptide” may not contain the first amino acid (D; aspartic acid) and the second amino acid (A; alanine) of the Aβ peptide. Accordingly, in this preferred embodiment, x is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide containing the second amino acid (A; alanine) of the Aβ peptide and y is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid) and the second amino acid (A; alanine) of the Aβ peptide.
In another embodiment, for the numerator (x), the epitope recognized by the antibody that is capable of binding with the detected “specific Aβ peptide” contains the third amino acid (E; glutamic acid) of the Aβ peptide and, for the denominator (y), the epitope recognized by the antibody that is capable of binding with the detected “specific Aβ peptide” may not contain the first amino acid (D; aspartic acid), the second amino acid (A; alanine) and the third amino acid (E; glutamic acid) of the Aβ peptide. Accordingly, in this preferred embodiment, x is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide containing the third amino acid (E; glutamic acid) of the Aβ peptide and y is the level of Aβ peptides capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine) and the third amino acid (E; glutamic acid) of the Aβ peptide.
The specific Aβ peptides detected in the numerator (x) of the ratio x/y are capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. Said specific Aβ peptides should thus at least contain a first amino acid (D; aspartic acid), second amino acid (A; alanine), and/or third amino acid (E; glutamic acid) of the Aβ peptides that is accessible for said antibody. “Accessible” means that said antibody is capable of forming an immunological complex with said epitope containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide.
The specific Aβ peptides detected in the denominator (y) of the ratio x/y are capable of forming an immunological complex with an antibody that recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. Said specific Aβ peptides thus lack an accessible first amino acid (D; aspartic acid), second amino acid (A; alanine), and/or third amino acid (E; glutamic acid) of the Aβ peptide. This lack of accessibility could be caused, for example, by the formation of aggregates or oligomers by said specific Aβ peptides wherein the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide are masked and thus not accessible anymore. Aggregated Aβ is identified as a mixture of oligomers in which the monomeric units are held together by non-covalent bonds. The lack of an accessible first amino acid (D; aspartic acid), second amino acid (A; alanine), and/or third amino acid (E; glutamic acid) of the Aβ peptide can also be caused by the presence of modifications on the N-terminal end of the Aβ peptide. Modification on the N-terminal end of the Aβ peptide may prevent the accessibility of the epitope comprising the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. An example of such modification includes (but is not limited to) acetylation.
The lack of an accessible first amino acid (D; aspartic acid), second amino acid (A; alanine), and/or third amino acid (E; glutamic acid) of the Aβ peptide can also be caused by the simple deletion of said N-terminal amino acids, resulting in N-terminally truncated Aβ peptides. N-terminally truncated Aβ peptides may start their amino acid sequence at amino acid 2 (A; Alanine), 3 (E; glutamic acid), 4 (F; phenylalanine), 5 (R; arginine), 6 (H; histidine), 7 (D; aspartic acid), 8 (S; serine), 9 (G; glycine), 10 (Y; tyrosine), 11 (E; glutamic acid), 12 (V; valine), 13 (H; histidine), 14 (H; histidine), 15 (Q; glutamine), 16 (K; lysine), or 17 (L; leucine) of the Aβ peptide. Some of the N-terminal truncated Aβ peptides that have been identified in human and animal cells, brains and/or CSF are shown in Table 2. In this specific embodiment, wherein the lack of an accessible epitope is caused by the deletion of one or more N-terminal amino acids, x may be the level of Aβ peptides that comprise said N-terminal amino acids of Aβ, also termed Aβ(1-C), wherein C means any possible C-terminal ending (see above). Preferably, C can be 42 and/or 43 and x is thus the level of Aβ(1-42) and/or Aβ(1-43) peptides. More preferably Aβ peptides ending at Ala42, i.e. Aβ(1-42), are detected and x is thus the level of Aβ(1-42) peptides. In the same specific embodiment (wherein the lack of an accessible epitope is caused by the deletion of one or more N-terminal amino acids), the specific Aβ peptides detected in the denominator (y) of the x/y ratio are then any Aβ peptide, starting at any amino acid of the Aβ peptide, referred to as Aβ(NC), wherein N means any possible N-terminal ending (i.e., amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 of Aβ). As indicated above, preferably, C can be 42 and/or 43 and y is the level of Aβ(N-42) and/or Aβ(N-43) peptides. More preferably, peptides ending at Ala42 of Aβ, i.e. Aβ(N-42), are detected. Accordingly, in these preferred embodiments, x is the level of Aβ(1-42) and/or Aβ(1-43) peptides and y is the level of Aβ(N-42) and/or Aβ(N-43) peptides. In another preferred embodiment, x is the level of Aβ(1-42) peptides and y is the level of Aβ(N-42) peptides. In another preferred embodiment, N is 11 and y is the level of Aβ(11-C) peptides. Preferably y is the level of Aβ(11-42) and/or Aβ(11-43) peptides. More preferably, y is the level of Aβ(11-42).
The term “level” or “levels”, as used in the present invention, refers to the amount of specific Aβ peptide present in the body fluid sample. The levels of specific Aβ peptide (x and y) obtained upon analyzing the body fluid samples and their ratio x/y will depend on the particular analytical protocol and detection technique that is used. Accordingly, those skilled in the art will understand that any laboratory, based on the present description, can establish a suitable reference range for the ratio x/y, characteristic (1) for the group of subjects that, at the time of sampling does not show any clinical signs of AD and that later develops AD, (2) for the group of subjects that, at the time of sampling does not show any clinical signs of AD and that later does not develop AD, (3) for the group of subjects that suffer from AD, (4) for the group of control subjects and (5) for the group of subjects that suffer from another dementia such as DLB. The ratio x/y obtained for the subject under diagnosis can then be compared with these reference ranges and, based on this comparison, a conclusion can be drawn as to which of the above groups the subject under diagnosis might belong.
The levels of the specific Aβ peptides may be determined by any method known to those skilled in the art. They can be identified by their structure, by partial amino acid sequence determination, by functional assay, by enzyme assay, by various immunological methods, or by biochemical methods such as capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyper diffusion chromatography, two-dimensional liquid phase electrophoresis (2D-LPE; Davidsson et al., 1999), or by their migration pattern in gel electrophoreses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used approach for separating proteins from complex mixtures (Patterson and Aebersold, 1995). It can be performed in a one- or two-dimensional (2D) configuration. Simultaneous analysis of different variants of Aβ peptides is provided by the Surface Enhanced Laser Desorption/Ionization (SELDI) ProteinChip™ Array (Ciphergen Biosystems Inc., Palo Alto, Calif., USA; Davies et al., 1999; Austen et al., 2000). In a preferred embodiment, the level of specific Aβ peptide is detected by an immunoassay. As used herein, an “immunoassay” is an assay that utilizes an antibody to specifically bind to the antigen (i.e., the specific Aβ peptide). The immunoassay is thus characterized by detection of specific binding of the specific Aβ peptide to antibodies. Immunoassays for detecting specific Aβ peptides may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (i.e., the specific Aβ peptide) is directly measured. In competitive assays, the amount of analyte (i.e., the specific Aβ peptide) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (i.e., the antibody) by the analyte (i.e., the specific Aβ peptide) present in the sample. In one competition assay, a known amount of the (exogenous) specific Aβ peptide is added to the sample and the sample is then contacted with the antibody. The amount of added (exogenous) specific Aβ peptide bound to the antibody is inversely proportional to the concentration of the specific Aβ peptide in the sample before the specific Aβ peptide is added. In one preferred “sandwich” assay, for example, the antibodies can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies (capturing antibodies) then capture the specific Aβ peptide of interest present in the test sample. Other immunological methods include, but are not limited to, fluid or gel precipitation reactions, immunodiffusion (single or double), agglutination assays, immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), Western blots, liposome immunoassays (LIA; Monroe et al., 1986), complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, or immunoPCR. An overview of different immunoassays is given in Wild (2001), Ghindilis et al. (2002) and Price and Newman (1997). Particularly advantageous are systems in which the levels of the different Aβ peptides, or the levels of the specific Aβ peptides, possibly together with other biological markers, can be detected simultaneously. In this multiparameter approach, antibodies may be coupled to microspheres or chips. An example of an immunoassay that provides for such simultaneous detection includes (but is not limited to) the xMap™ technology (Luminex 100 IS, Austin, Tex., USA).
In a preferred embodiment, the level of the specific Aβ peptide is determined by an immunoassay comprising at least the following steps:
In the methods of the present invention, the ratio x/y is thus determined immunologically making use of two antibodies (a set of antibodies), i.e. a first antibody (detecting x) that specifically recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide and a second antibody (detecting y) that recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide.
In another preferred embodiment, the specific Aβ peptide can be detected by a sandwich ELISA comprising the following steps:
Advantageously, the detector antibody itself carries a marker or a group for direct or indirect coupling with a marker.
In the method of the present invention, the ratio x/y can thus be determined immunologically making use of two capturing antibodies (or a set of antibodies), i.e. a first (capturing) antibody (detecting x) that specifically recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide and a second (capturing) antibody (detecting y) that recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. In a preferred embodiment of the present invention, the first antibody specifically recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid) of the Aβ peptide and the second antibody recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. In another preferred embodiment, the first antibody is the monoclonal antibody 3D6, BAN-S0, or Anti-N1(D) and the second antibody recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide. In another preferred embodiment of the present invention, the first antibody specifically recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide and the second antibody recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid) of the Aβ peptide. In another preferred embodiment of the present invention, the first antibody specifically recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid) of the Aβ peptide and the second antibody recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid) of the Aβ peptide. In another preferred embodiment of the present invention, the first antibody is the monoclonal antibody 3D6, BAN-50, or Anti-N1(D) and the second antibody recognizes an epitope of the Aβ peptide not containing the first amino acid (D; aspartic acid) of the Aβ peptide. In another preferred embodiment of the present invention, the first antibody specifically recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid), the second amino acid (A; alanine), and/or the third amino acid (E; glutamic acid) of the Aβ peptide and the second antibody recognizes an epitope of the Aβ peptide different from the 3D6, BAN-50, and/or Anti-N1(D) epitope. In another preferred embodiment of the present invention, the first antibody specifically recognizes an epitope of the Aβ peptide containing the first amino acid (D; aspartic acid) of the Aβ peptide and the second antibody recognizes an epitope of the Aβ peptide different from the 3D6, BAN-50, and/or Anti-N1(D) epitope. In another preferred embodiment of the present invention, the first antibody is the monoclonal antibody 3D6, BAN-50 or Anti-N1(D) and the second antibody recognizes an epitope of the Aβ peptide different from the 3D6, BAN-50, and/or Anti-N1(D) eptiope. Any antibody that specifically recognizes the specific Aβ peptides under examination can be used in the above method. Examples of antibodies known in the art are provided in Table 1.
The detector antibody to be used in the sandwich ELISA as discussed above, can be any antibody that recognizes an epitope on the Aβ peptide or on the Aβ peptide-capturing antibody complex, not masked by the capturing antibody. Examples of antibodies that can be used as detector antibody are given in Table 3. In a preferred embodiment, Aβ peptides ending at Ala42 (i.e. Aβ42) or Thr43 (i.e. Aβ43) are detected with an antibody that specifically recognizes Aβ42 and/or Aβ43. In another preferred embodiment, Aβ peptides ending at Ala42 (i.e. Aβ42) are detected.
It is evident that, in the sandwich ELISA of the present invention, the capturing and detector antibodies could change place. Thus, the first and second antibody as discussed above, could also be used as detector antibody, when, as a capturing antibody, an antibody is used that recognizes an epitope on the Aβ peptide different from the epitope recognized by these first and/or second antibody.
The antibodies as discussed above can be used in the preparation of a diagnostic kit for use in the methods of the present invention. Accordingly, the present invention relates to a first antibody and a second antibody (a set of antibodies) as discussed above, for the manufacture of a diagnostic kit for determining whether a subject has a likelihood to develop AD, for the diagnosis of a subject suffering from AD and/or for the differential diagnosis of a subject suffering from AD versus a subject suffering from another dementia such as DLB.
As used in the present invention, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or dimer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The terms “variable light chain (VL)” and “variable heavy chain (VH)” refer to these variable regions of the light and heavy chains respectively. Optionally, the antibody or the immunological portion of the antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
Antibodies used in the present invention include, but are not limited to polyclonal, monoclonal, bispecific, human, humanized, or chimeric antibodies, single variable fragments (ssFv), single chain fragments (scFv), Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies, and epitope-binding fragments of any of the above, provided that they retain the original binding properties. Also mini-antibodies and multivalent antibodies such as diabodies, triabodies, tetravalent antibodies and peptabodies can be used in a method of the invention. The preparation and use of these fragments and multivalent antibodies has been described extensively in International Patent Application WO 98/29442. The immunoglobulin molecules of the invention can be of any class (i.e. IgG, IgE, IgM, IgD, and IgA) or subclass of immunoglobulin molecule.
The specific Aβ peptides detected in the present invention can be used as an immunogen to generate the antibodies used in the invention which specifically bind such an immunogen. Various host animals can be immunized for injection with said specific Aβ peptides, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to enhance the immunological response, depending on the host species, including but not limited to complete or incomplete Freund's adjuvant, a mineral gel such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyol, a polyanion, a peptide, an oil emulsion, keyhole limpet hemocyanin, dinitrophenol, or an adjuvant such as BCG (bacille Calmette-Guerin), or corynebacterium parvum. For the preparation of monoclonal antibodies, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. Hyperimmunization of an appropriate donor, generally a mouse, with the antigen is undertaken. Isolation of splenic antibody producing cells is then carried out. These cells are fused to a cell characterized by immortality, such as a myeloma cell, to provide a fused cell hybrid (Hybridoma) which can be maintained in culture and which secretes the required monoclonal antibody. The cells are then cultured in bulk and the monoclonal antibodies harvested from the culture media for use. Specific techniques include but are not limited to the hybridoma technique developed by Kohler and Milstein (1975), the human B-cell hybridoma technique (Kozbor et al., 1983), or the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985). Screening for the desired antibody can be done by techniques known in the art, such as ELISA. For selection of an antibody that specifically binds a specific Aβ peptides, but that does not specifically bind another protein, selection can be made on the basis of positive binding to the first and the lack of binding to the second.
While various antibody fragments are defined in terms of enzymatic digestion of an intact antibody with papain, pepsin or other proteases, one skilled in the art will appreciate that such antibody fragments as well as full size antibodies may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibodies and antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. The term “humanized antibody” means that at least a portion of the framework regions of an immunoglobulin is derived from human immunoglobulin sequences. The humanized versions of the mouse monoclonal antibodies can, for example, be made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (Queen et al., 1989; WO 90/07861). Alternatively, the monoclonal antibodies used in the method of the invention may be human monoclonal antibodies. Human antibodies can be obtained, for example, using phage-display methods (WO 91/17271; WO 92/01047). In these methods, libraries of phage are produced in which members display different antibodies on their outersurfaces. Antibodies are usually displayed as Fv or Fab fragments. Human antibodies against specific Aβ peptides can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus (WO 93/12227; WO 91/10741). Human antibodies can be selected by competitive binding experiments, or otherwise to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using specific Aβ peptides as an affinity reagent. Monoclonal antibodies can be obtained from serum according to the technique described in WO 99/60846. Also useful in the above methods might be the heavy chain variable domains (VHH) produced as part of the humoral immune response of camelids. Recombinant VHH selected from ‘camelised’ human VH libraries could consitute excellent ligands for the detection of the specific Aβ peptides of the present invention (Spinelli et al., 2000; Muyldermans, 2001; Cortez-Retamozo et al., 2002).
The antibodies used in the methods of the present invention may be labeled by an appropriate marker or label. The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and, in general, almost any label useful in such methods can be applied to the methods of the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, radiological or chemical means. Useful labels in the present invention include but are not limited to magnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g. fluorescein isothiocyanate, Texas Red, rhodamine, phycoerythrin, Alexa 532, cyanine 3), radiolabels (e.g. 3H, 125I, 35S, 14C, or 32P), enzymes (e.g. horseradish peroxidase, alkaline phosphatase, and others commonly used in an ELISA), and colorimetric labels such as colloidal gold, colored glass, or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, the available instrumentation and disposal provisions. Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the antibody. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, a haptenic or antigenic compound can be used in combination with an antibody. The antibodies can also be conjugated directly to signal generating compounds; for example, by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umberlliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, for example, luminol. A review of other labeling or signal-producing system is available in U.S. Pat. No. 4,391,904.
Means for detecting labels are well known in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of a photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzyme labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected simply by observing the color associated with the label.
Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
The present invention also provides diagnostic kits comprising the antibodies as referred to above. The invention thus provides a diagnostic kit for determining whether a subject has a likelihood to develop AD, for the diagnosis of a subject suffering from AD and/or for the differential diagnosis of a subject suffering from AD versus a subject suffering from another dementia such as DLB, comprising at least a first antibody and a second antibody (a set of antibodies) as discussed above.
A preferred kit for carrying out the methods of the present invention comprises:
The present invention thus provides a first and second antibody (a set of antibodies) or a diagnostic kit, as defined above, for use in the determination of whether a subject has a likelihood to develop AD, for the diagnosis of a subject suffering from AD and/or for use in the differential diagnosis of a subject suffering from AD versus a subject suffering from another dementia such as DLB.
In the methods of the present invention, the detection of at least the ratio x/y, may optionally be combined with the detection of one or more additional known biomarkers for neurological diseases, including but not limited to other Aβ peptides, tau, phospho-tau, synuclein, Rab3a, cytokines, glutamine synthase (GS) and neural thread protein. Combination of relevant biological markers may increase the sensitivity and specificity of the diagnosis. The methods of the invention can also be used for further confirmation of a diagnosis previously made with one or more other biological markers.
The methods, diagnostic kits and/or set of antibodies of the present invention can also be used for monitoring the effect of therapy administered to a subject, also called therapeutic monitoring or treatment follow up, and patient management. Accordingly, the present invention is also related to the methods as described above for use in the treatment follow up of a subject that has a likelihood to develop AD or of a subject that is diagnosed as suffering from AD. Changes in the x/y ratio can be used to evaluate the response of a subject to drug treatment. In this way, new treatment regimes can also be developed by examining the x/y ratio in a subject. The method of the present invention can thus assist in monitoring a clinical study, for example, for evaluation of a certain therapy for memory impaired subjects, subjects with MCI or subjects suffering from AD. In this case, a chemical compound is tested for its ability to normalize the x/y ratio in a subject diagnosed as developing or suffering from AD.
The methods of the present invention can also be used in animal or cellular models, for example, for drug screening. The animal model on which the method of the present invention can be applied can be any model of an animal in which the body control system is directed by CNS. The animal thus may belong to the Platyhelminthes, Aschelminthes, Annelida, Arthropoda, Mollusca, Echinodermata, Acrania, Cyclostomata, Chondrichthyes, Osteichthyes, Amphibia, Reptilia, Aves, and Mammalia. In a preferred embodiment, the animal in the animal model is a mouse, a rat, a monkey, a rabbit, a worm, a fly, a zebrafish, a pufferfish or C. elegans. In another embodiment, the animal is a transgenic animal, possibly modified by one or more determinants causing AD. Cellular models on which the method of the present invention can be applied can be any cell line in which APP is expressed. Examples include APP expressing primary cultures of neurons as described by De Jonghe et al. (2001), CHO (Chinese Hamster Ovarian) cells transfected with human wild type or mutant APP and human neuroblastoma cells (SKNSH-SY5Y) transfected with human wild type or mutant APP as described in WO 02/37118.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of stated integers or steps but not to the exclusion of any other integer or step or group of integers or steps.
The present invention will now be illustrated by reference to the following examples that set forth particularly advantageous embodiments. However, it should be noted that these examples are illustrative and can not be construed as to restrict the invention in any way.
1. Determination of the Binding Specificities of Monoclonal Antibodies 3D6, 6E10 and 4G8 in ELISA
Three β-amyloid antibodies, 3D6 (Elan Pharmaceuticals, South San Francisco, Calif., USA), 6E10 and 4G8 (Signet Laboratories, Dedham, Mass., USA) were tested in ELISA for immunological binding with different β-amyloid peptides truncated at their N-terminal end: Aβ(1-42), Aβ(2-42), Aβ(3-42), Aβ(4-42), Aβ(5-42), Aβ(8-42), Aβ(9-42). Synthetic peptides were obtained from Bachem (Heidelberg, Germany), Neosystems (Strasbourg, France), or AnaSpec (San Jose, Calif., USA). The ELISA format and its characteristics have previously been described in detail (Vanderstichele et al., 2000). In short, plates were pre-coated with the 3D6, 6E10 or 4G8 monoclonal antibody, specific for the N-terminus of Aβ42, as capturing antibody. To each well, 100 μl of blank or peptide sample were added and incubated for three hours. After several wash steps, the plates were incubated for 1 h with the biotinylated monoclonal antibody 21F12, specific for the carboxy terminus of Aβ42. Biotinylated antibodies were detected via peroxidase-labeled streptavidin. After the final washing step, substrate was added and the reaction was stopped after 30 min by adding 0.2 N sulphuric acid. The optical density (OD) was measured at 450 nm.
The reactivity of 3D6, 6E10 and 4G8 with the different N-terminally truncated Aβpeptides is shown in
2. Determination of the Binding Specificities of Monoclonal Antibodies 3D6, 6E10 and 4G8 in a Multiparameter Immunoassay
The three β-amyloid antibodies, 3D6, 6E10 and 4G8 were tested in a multiparameter assay for immunological binding with Aβ(1-42) and Aβ(2-42). Synthetic peptides were obtained from Bachem (Heidelberg, Germany), Neosystems (Strasbourg, France), or AnaSpec (San Jose, Calif., USA).
The xMAP™-technology (Luminex, Austin, Tex., USA) was used to design a multiparametric bead-based assay. 3D6, 6E10, and 4G8 were covalently coupled onto carboxylated microsphere sets according to a modified protocol supplied by the manufacturer. In short, a mixture of water-soluble 1-ethyl-3-(3-dimethyl-laminopropyl)-carbodiimide hydrochloride (EDC; Pierce Chemicals, Erembodegem, Belgium) and N-hydroxy-sulfosuccinimide (Sulfo-NHS; Pierce Chemicals, Erembodegem, Belgium) was used to activate the free carboxyl groups on the beads. The amino groups of the antibodies were subsequently covalently bound to the carboxy groups of the microspheres. The antibody-coupled beads were counted using a haemocytometer. Coupled microspheres were stored in the dark at 2-8° C.
All incubations were performed at room temperature (25° C.). A ninety-six well filter plate (Millipore Corporation, Bedford, Mass., US) was first pretreated with 250 μl wash buffer (PBS, 0.05% Tween20). The wash buffer was removed from the plates with a vacuum manifold (Millipore Corporation, Brussels, Belgium). The coupled microspheres were allowed to eliquilibrate at room temperature for at least 15 min, after which 100 μl of sonicated microspheres (30.000 beads/ml for each parameter) were added to the filterplate, after which the plates were covered with aluminium foil. After aspiration of the buffer, 50 μl of the biotinylated detector antibody 21F12 (Innogenetics N.V., Gent, Belgium) and 50 μl of the peptide sample were added to the wells. Incubation was performed overnight (16 hours) by shaking at room temperature on an orbital plate shaker at 1000 rpm. The wells were washed three times by aspiration with 300 μl of wash buffer and incubated with 100 μl of Streptavidin-phycoerythrine (Streptavidin-PE; Caltag, Burlingame, Calif., US; Sanbio, Uden, the Netherlands) for 60 min on a plate shaker. The wells were again washed three times by aspiration with wash buffer. Finally, the microspheres were redissolved in PBS. The fluorescence intensity was measured in the Luminex 100 instrument using software version 2.1 for analysis.
The reactivity of 3D6, 6E10 and 4G8 with the Aβ(1-42) and Aβ(2-42) peptides is shown in
1. Subjects
A study was carried out based on CSF samples archived at the Sahlgren's University Hospital, Göteborg, Sweden, including 166 subjects. For 12 CSF samples, not all parameters were determined. Exclusion of these partial results did not affect the final analysis. The results of 154 samples are discussed here, including the following patient groups: 18 patients with moderate AD (modAD), 21 patients with severe AD (sevAD), 20 patients with mild AD (mildAD), 39 patients with cognitive impairment, 12 patients with dementia with Lewy bodies (DLB), 15 patients with Parkinson's disease (PD) and 29 control subjects (C). All patients with AD satisfied the NINCDS-ADRDA criteria (McKhann et al., 1984). For the patient group with cognitive impairment, no symptom other than memory impairment was reported or identified and none of these patients fulfilled the DSM-IV criteria for dementia. Within a follow up period of 5 years, 14 patients with cognitive impairment had progressed to AD (Cog-AD) whereas in 25 patients with cognitive impairment, the memory problems did not develop into AD (Cog). DLB was diagnosed according to the Consensus guidelines for the clinical and pathological diagnosis of dementia with Lewy bodies (McKeith et al., 1996). PD patients were included according to Langston et al., (1992). The control group (C) consisted of individuals without histories, symptoms, or signs of psychiatric or neurological disease, malignant disease, or systemic disorders (e.g., rheumatoid arthritis, infectious disease).
The Ethics Committees of the University of Göteborg, Sweden, approved the study. All patients (or their nearest relatives) and controls gave informed consent to participate in the study, which was conducted according to the provisions of the Declaration of Helsinki.
2. Sampling
CSF samples were taken using atraumatic cannulas placed in the L3/L4 or L4/L5 invertebral space of the subject. 12 ml was collected in sterile polypropylene tubes and gently mixed. The CSF was centrifuged for 10 minutes at 4000 g. Samples were sent to the Clinical Neurochemistry Laboratory at Sahlgrens's University Hospital in Mölndal Sweden. After arrival, samples were aliquoted and kept frozen at −80° C. Samples were kept without being thawed and refrozen. In the native CSF, determination of routine chemical parameters was performed, including leucocyte and erythrocyte cell counts as well as glucose and lactate measurements, total protein content, CSF-serum ratios of albumin and immunoglobulin G, and a screening for oligoclonal bands. CSF samples were not included in the study if they contained more than 500 red blood cells per μL.
3. Immunoassay
The levels x and y of specific Aβ peptides in the CSF samples were determined using the xMap™ technology (Luminex 100IS, Austin, Tex., US). As capturing antibody, the monoclonal antibody 3D6 (Elan Pharmaceuticals, South San Francisco, Calif., US) was used for the detection of x and the monoclonal antibodies 6E10 and 4G8 (Signet Laboratories, Dedham, Mass., US) for the detection of y. The monoclonal antibody 21F12 (Innogenetics N.V., Gent, Belgium) was used as detector antibody. The multiparameter assay was carried out as described above. Luminex units were converted to ELISA pg peptide equivalents/ml using synthetic amyloid(1-42) peptide as a standard and a sigmoidal fit as a curve fitting model.
4. Statistical Analysis
Data analysis was based on graphical representation by means of box plots. These plots represent the median values and the 25%-75% range that covers the middle 50% of the data points for every variable. Additional support for the capacity of a variable to discriminate between 2 groups is provided by a formal Mann-Whitney U test. This test assesses the hypothesis that the sum of the ranks of data points in each group is the same. Significant p values (<0.05) reject this hypothesis and thus provide strong evidence for the discriminating ability of a variable between the 2 groups. P values <0.10 suggest a trend for the discriminating ability of a variable between the 2 groups.
5. Discrimination of Memory-Impaired Subjects that Will Progress to AD from Patients with Prolonged Memory Problems
In
In
6. Discrimination of Subjects Suffering from AD from Subjects Suffering from DLB
In FIGS. 2 ([x]3D6), 3 ([y]6E10), and 4 ([y]4G8), the median and the 25%-75% interval are shown for the levels x and y of specific Aβ peptides in the CSF samples of the different subject groups using 3D6, 6E10, and 4G8 as capturing antibody, respectively. While a clear decrease can be observed in the level of Aβ peptides binding 3D6 between patients who suffer from any form of AD (ModAD, SevAD, MildAD) compared to control subjects (C), to patients with PD (PD) or to patients suffering from memory impairment (Mem, Mem-AD), the levels x or y did not provide for the discrimination between patients suffering from AD versus patients suffering from DLB. In the comparison of all AD (ModAD, SevAD, and MildAD pooled) to DLB the p values of the Mann-Whitney U test were 0.16, 0.68, and 0.79 for [x]3D 6, [y]6E10 and [y]4G8, respectively.
In FIGS. 5 ([x]3D6/[y]6E10) and 6 ([x]3D6/[y]4G8), the median and the 25%-75% interval are shown for the ratios of x/y in the CSF samples of the different subject groups. For this ratio, a clear decrease can be observed for patients suffering from any form of AD (ModAD, SevAD, MildAD) compared to patients suffering from DLB. The p values of the Mann-Whitney U test were 0.098 and <0.001 for [x]3D6/[y]6E10 and [x]3D6/[y]4G8, respectively. This shows that the ratio of x/y allows discrimination between subjects suffering from AD versus subjects suffering from DLB.
1. Subjects
CSF samples from patients with AD (n=22) and non-demented controls (n=20) were provided by the Sahlgren's University Hospital, Göteborg, Sweden. They were collected for the purpose of routine diagnostic procedure from patients diagnosed according to the generally accepted criteria of AD, ICD-10 (World Health Organization, 1992) and National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related disorder Association (NINCDS-ADRDA criteria; McKann et al., 1984). The control group consisted of individuals without histories, symptoms, or signs of psychiatric or neurological disease.
The Ethics Committees of the Universities of Göteborg, Sweden, approved the study. All patients (or their nearest relatives) and controls gave informed consent to participate in the study, which was conducted according to the provisions of the Declaration of Helsinki.
2. Analysis of the Levels x and y of Specific Aβ Peptides by Multiparameter Immunoassay
The levels x and y of specific Aβ peptides in the CSF samples were determined using the xMAp™-technology (Luminex, Austin, Tex., USA). As capturing antibody, the monoclonal antibody 3D6 was used for detection of x and the monoclonal antibody 4G8 for detection of y. The monoclonal antibody 21F12 was used as detector antibody. The multiparameter assay was carried out as described above.
Data analysis was based on graphical representation by means of box plots. These represent the median values and the 25%-75% range that covers the middle 50% of the data points for every variable. Additional support for the capacity of a variable to discriminate between the 2 groups (AD and controls) was provide by the Student-Newman-Keuls test for pairwise comparisons. A significant p value (<0.05) provides strong support for the discriminating ability of a variable between AD and control patients.
In
In
3. Analysis of the Levels x and y of Specific Aβ Peptides by Surface-Enhanced Laser Desorption/Ionization Technology
Surface-enhanced laser desorption/ionization (SELDI) technology was applied to analyze and compare the patterns of Aβ(42) peptide in CSF of 22 individual CSF samples from patients with AD (n=22) and non-demented controls (n=20). The CSF samples were analyzed on the SELDI-TOF (PBS IIc) for the presence of specific Aβ(42) peptides. To allow this, the Aβ peptides were immunologically bound to a ProteinChip by the following immuno-array preparation protocol. A monoclonal antibody (4D7A3; Innogenetics Cat. No. BR032D) directed towards C-terminus of β-amyloid(42) peptide was covalently linked to the PS10 ProteinChip by applying 1 μg 4D7A3 on the array-spot and incubation in a humidity chamber (3 h, RT) to allow covalent binding to the PS10 ProteinChip array. The arrays were washed twice with PBS/0.1% Triton X-100. Additional washing was performed 2× with PBS (pH 8.0). The arrays were blocked with 0.5 M Tris pH 8.0 (during 2 h, RT). The arrays were again washed twice with PBS/0.1% Triton X-100. 100 μl CSF in 0.1M urea/0.1% CHAPS was loaded on a spot using the ProteinChip bioprocessor and incubated by constant shaking, overnight at 4° C. The arrays were again washed twice with PBS/0.1% Triton X-100 and two additional washes with 50 mM Hepes pH 8.0. On the air-dried array spots α-cyano-4-hydroxycinnamic acid in 50% ACN/50% TFA was applied and mass analysis was performed. The same immuno-array was prepared with antibody 31 D11 as control antibody to obtain a spectrum without any Aβ peptide (
External calibration was performed with Dynorphorin (Mr=2147,50 Da), human ACTH(1-24) (2933,50), Bovine insulin beta-chain (3495,94), and human insulin (5807,65). On this basis mass accuracy was calculated for the Aβ(42) peptide peak with theoretical mass of 4514,1 Da. The m/z value measured by SELDI-TOF was 4512,069 Da (STDEV 1,193456, % CV 0,02645) giving the accuracy for this experiment of 450 ppm. Taking this mass accuracy of the SELDI-TOF (450 ppm) into a account, and by using complementary analysis of relevant synthetic β-amyloid peptides, novel N-terminally truncated β-amyloid peptides were assigned on the basis of their molecular masses as follows: 1-42, 11-42, 8-42, 5-42 and 3-42 (
In order to improve measurements, the same samples were exposed to the high-laser intensity and for several samples more accurate data were obtained. For the calibration purposes 7 fmol 9-42 β-amyloid peptide (AnaSpec San Jose, Calif.; cat. No. 60084-1) and 6 fmol bovine insulin (Ciphergene Biosystems Fremont, Calif.) were applied and were used for data calibration. In
Tables
p: pyroglutamate;
C: C-terminal end of Aβ is not defined.
ox: oxidized form.
| Number | Date | Country | Kind |
|---|---|---|---|
| 03447120.1 | May 2003 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| 60477621 | Jun 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10848686 | May 2004 | US |
| Child | 11810204 | Jun 2007 | US |
